CN112852900A - Pretreatment method and enzymolysis method of lignocellulose raw material - Google Patents
Pretreatment method and enzymolysis method of lignocellulose raw material Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
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- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
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Abstract
The invention provides a pretreatment method and an enzymolysis method of a lignocellulose raw material. The pretreatment method comprises the following steps: step S1, swelling the lignocellulose raw material by using an alkaline salt solution to obtain a swelling product; and step S2, introducing oxygen into the swelling product for oxidation treatment to obtain a pretreated product. The lignocellulose raw material is swelled by adopting the alkaline salt solution, the swelled lignocellulose is soaked, and then the swelled product is placed in an oxygen environment for oxidation treatment, so that most of lignin and partial hemicellulose are removed under the condition of keeping more cellulose, namely, the decomposition efficiency of the lignocellulose and the hemicellulose is improved by pretreatment, the subsequent enzymolysis is facilitated, and the enzymolysis saccharification efficiency and the reducing sugar yield are greatly improved.
Description
Technical Field
The invention relates to the technical field of renewable energy sources, in particular to a pretreatment method and an enzymolysis method of a lignocellulose raw material.
Background
Due to the rapid depletion of petroleum feedstocks and the serious environmental problems caused by the burning of fossil fuels, lignocellulose, an alternative energy source, has attracted attention for the production of renewable biofuels and value-added chemicals. The lignocellulose raw material has the advantages of being rich in variety, wide in source, low in price, easy to obtain and the like, and the source of the lignocellulose raw material mainly comprises cellulose resources in agricultural wastes, forestry wastes, energy crops and municipal wastes. The quantity of the agricultural wastes is huge, and the reasonable and resource utilization of the agricultural wastes is helpful for relieving the impact caused by the shortage of fossil fuels and solving the problem of recycling the agricultural wastes.
Lignocellulose mainly comprises three components of cellulose (35-50%), hemicellulose (20-35%) and lignin (5-30%). Wherein the cellulose and the hemicellulose mainly comprise glucose and xylose, and can be further used for producing chemicals with high added values through chemical and biological conversion after being degraded into monosaccharide through hydrolysis reaction. However, in plant tissues lignin is covalently bound to hemicellulose and entraps cellulose therein, forming a strong natural barrier against microbial and chemical degradation. Therefore, effective pretreatment means is needed to break the structure of lignocellulose, remove lignin or hemicellulose, reduce the crystallinity of the substrate or increase the specific surface area of the substrate, thereby improving the degradation rate of the substrate and the yield of reducing sugar in the subsequent enzymatic hydrolysis process.
The currently available pretreatment methods mainly include: physical methods (mechanical pulverization, pyrolysis, steam explosion, liquid hot water treatment), chemical methods (alkali treatment, ammonia treatment, organic solvent method), physicochemical methods, biological methods, and combination treatment methods. The alkaline pretreatment is one of the most widely applied pretreatment methods at present, and an alkaline reagent can weaken hydrogen bonds between cellulose and hemicellulose, saponify ester bonds between the hemicellulose and lignin, enable a lignocellulose raw material to generate a swelling effect, and separate the lignin from carbohydrates, so that the lignin is removed to achieve the purpose of improving the hydrolysis performance of a substrate. The conventional alkaline pretreatment is mainly ammonia cycle percolation (ARP), ammonia explosion (AFEX), ammonia Soak (SAA), Low Liquid Ammonia (LLA) and Low Moisture Anhydrous Ammonia (LMAA) and by using other strong alkaline solutions, such as NaOH or Ca (OH)2And the like, the alkaline agent is difficult to recover and the corrosion of equipment is large. The pretreatment of the oxidant can effectively decompose lignin and dissolve a large amount of hemicellulose, so that the accessibility of the cellulose is improved, and the commonly used oxidant mainly comprises hydrogen peroxide, peroxyacetic acid and the like.
Disclosure of Invention
The invention mainly aims to provide a pretreatment method and an enzymolysis method of a lignocellulose raw material, so as to solve the problem of low effective decomposition rate of cellulose in the prior art.
In order to achieve the above object, according to one aspect of the present invention, there is provided a pretreatment method of a lignocellulosic feedstock, the pretreatment method comprising: step S1, swelling the lignocellulose raw material by using an alkaline salt solution to obtain a swelling product; and step S2, introducing oxygen into the swelling product for oxidation treatment to obtain a pretreated product.
Further, step S2 includes: step S21, placing the swelling product in a closed reaction kettle, introducing oxygen into the closed reaction kettle, and controlling the pressure of the introduced oxygen to be 0.2-3.0 MPa, preferably 0.2-2.0 MPa; and step S22, heating and oxidizing the swelling product to obtain a pretreated product.
Further, the temperature-raising oxidation treatment includes: the temperature of the swelling product is raised to 100-160 ℃, and the swelling product is oxidized for 0.3-2 h at the temperature.
Further, after the temperature-rising oxidation treatment and before the pretreatment product is obtained, the pretreatment method further comprises the following steps: performing water bath cooling on the product subjected to temperature rise and oxidation treatment, preferably performing ice water bath cooling to obtain a cooled product; and filtering and washing the cooled product to obtain a pretreated product.
Further, the mass volume concentration of the lignocellulose raw material is 5-20% of the substrate concentration.
Further, before step S1, the preprocessing method further includes: the lignocellulosic feedstock is comminuted into particles, preferably having a particle size of from 0.1cm to 1.5cm, more preferably from 0.3cm to 1.5 cm.
Further, the solid-liquid mass ratio of the lignocellulose raw material to the alkaline salt solution is as follows: 1: 5-1: 20, preferably, the temperature of the swelling treatment is 30-50 ℃, and more preferably, the time of the swelling treatment is 1-4 h.
Further, the alkaline salt solution is a solution of a weakly alkaline salt; preferably, the weakly basic salt is sodium phosphate, sodium acetate, sodium carbonate, sodium sulfide, sodium hypochlorite or sodium silicate.
Further, the concentration of the alkaline salt solution is 2 wt% to 20 wt%.
Further, the lignocellulose raw material is corn stalk, rice straw, wheat straw, spring bamboo shoot skin or bagasse.
In order to achieve the above object, according to one aspect of the present invention, there is provided an enzymatic hydrolysis method of a lignocellulosic feedstock, the enzymatic hydrolysis method comprising: pretreating the lignocellulose raw material by any one of the pretreatment methods to obtain a pretreated product; and carrying out enzymolysis on the pretreated product to obtain reducing sugar.
Further, performing enzymolysis on the pretreated product by using a complex enzyme, wherein the complex enzyme is a mixture of cellulase and xylanase, and preferably, in the complex enzyme, the enzyme activity ratio of the cellulase to the xylanase in unit volume is as follows: the cellulase is xylanase, namely 1FPU:1FXU, wherein the FPU is a filter paper enzyme activity unit of the cellulase, and the FXU is an enzyme activity unit of the xylanase; more preferably, the amount of the complex enzyme is 10FPU/(g substrate) to 30FPU/(g substrate).
Further, the enzymolysis is carried out under an acetic acid-sodium acetate buffer solution with the temperature of 50 ℃ and the pH value of 4.8, and the preferable time of the enzymolysis is 72 to 96 hours.
By applying the technical scheme of the invention, the lignocellulose raw material is firstly subjected to swelling treatment by adopting the alkaline salt solution, the swollen lignocellulose is soaked, and then the swelling product is placed in an oxygen environment for oxidation treatment, so that most of lignin and part of hemicellulose are removed under the condition of keeping more cellulose, namely the decomposition efficiency of the pretreatment on the lignocellulose and the hemicellulose is improved, the subsequent enzymolysis is facilitated, and the enzymolysis saccharification efficiency and the reducing sugar yield are greatly improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows the effect of different substrate concentrations on the solid recovery, reducing sugar yield, glucose yield and lignin removal rate of corn stover samples.
FIG. 2 shows the material balance relationship between corn stalks pretreated with sodium phosphate and hydrolyzed with enzyme.
FIG. 3 shows the enzymatic hydrolysis of corn stover at different degrees of comminution.
FIG. 4 shows the 72 hour enzymatic reducing sugar yield for pretreatment of substrates at various substrate loadings.
FIG. 5 is a Scanning Electron Microscope (SEM) image of corn stover before and after sodium phosphate pretreatment.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background art, the effective decomposition rate of lignocellulose by the pretreatment method of the lignocellulose raw material in the prior art is low, so that the yield of reducing sugar after enzymolysis is relatively low, and in order to further improve the decomposition efficiency of lignocellulose and increase the yield of corresponding reducing sugar, the inventor of the present application has conducted various comparison studies on the existing pretreatment method, and found that the existing pretreatment method mostly adopts a single treatment method, such as the most widely used alkaline pretreatment method. Although there are a few reports of pretreatment by combination, most of the combinations are pretreated in the form of a combined solvent, for example, a lignin fiber raw material is pretreated by a combined solvent formed by mixing an alkaline pretreatment solution and an oxidative pretreatment solution (hydrogen peroxide), and the treatment can improve the decomposition efficiency of lignocellulose and hemicellulose to some extent, but the decomposition efficiency is still to be improved. Further, the inventors have further analyzed that, in the case where the alkaline solvent and the oxidizing solvent act simultaneously on the lignocellulosic raw material during the treatment, the decomposition efficiencies of the both are interfered with each other for some unknown reason, and the respective advantages cannot be fully exerted, so that the improvement of the decomposition efficiency is limited. To verify this, the inventors verified that the decomposition efficiency was not much different from that of the combined solvent by successively treating the basic solvent and the oxidizing solvent. The inventor considers the aspects of different forms of the solvent again, particularly the oxidation pretreatment process, and finds that the liquid oxidation treatment is changed into the gaseous oxidation treatment, the contact efficiency and the reaction efficiency of the alkaline pretreatment product under the gaseous environment are higher, the decomposition of the lignocellulose raw material is more thorough, and the yield of the reducing sugar obtained by the subsequent enzymolysis is higher.
On the basis of the research results, the applicant proposes an improvement scheme of the application. In an exemplary embodiment of the present application, there is provided a method of pretreating a lignocellulosic feedstock, the method comprising: step S1, swelling the lignocellulose raw material by using an alkaline salt solution to obtain a swelling product; and step S2, introducing oxygen into the swelling product for oxidation treatment to obtain a pretreated product.
The oxidation effect of oxygen is stronger, and the effect of removing lignin is better. Since oxygen in alkaline solutions can attack the more electron-rich radical region of the substrate (lignocellulose) in the form of free radicals, a series of changes occur during the reaction, as shown in the following reaction scheme:
wherein, the diffusion range of the superoxide radical in the liquid is larger than that of other radicals such as hydroxyl, so that the lignin can be more effectively removed. In the prior method of combining alkaline salt with hydrogen peroxide, the hydrogen peroxide cannot be recycled after being used once, the reagent cost is high, and the difficulty of subsequent wastewater treatment is increased.
Therefore, according to the pretreatment method, the lignocellulose raw material is subjected to swelling treatment by adopting the alkaline salt solution, the swollen lignocellulose is soaked, and then the swollen product is placed in the oxygen environment for oxidation treatment, so that most of lignin and part of hemicellulose are removed under the condition of keeping more cellulose, namely, the decomposition efficiency of the pretreatment on the lignocellulose and the hemicellulose is improved, the subsequent enzymolysis is facilitated, and the enzymolysis saccharification efficiency and the reducing sugar yield are greatly improved.
In the step of the oxygen oxidation treatment, the specific equipment for the oxidation treatment can be reasonably selected according to actual production conditions. In a preferred embodiment, step S2 includes: step S21, placing the swelling product in a closed reaction kettle, introducing oxygen into the closed reaction kettle, and controlling the pressure of the introduced oxygen to be 0.2-3.0 MPa, preferably 0.2-2.0 MPa; and step S22, heating and oxidizing the swelling product to obtain a pretreated product. And the closed reaction kettle is adopted for oxidation treatment, so that the oxidation treatment effect is improved. And the pressure of oxygen in the closed reaction kettle is controlled within the pressure range, so that the oxidation efficiency is high, and the decomposition of lignocellulose and hemicellulose is facilitated.
In a preferred embodiment, the elevated temperature oxidation process comprises: the temperature of the swelling product is raised to 100-160 ℃, and the swelling product is oxidized for 0.3-2 h at the temperature. The selection of the reaction temperature and the reaction time is mainly found in the practical research process, when the temperature is lower than the temperature or the reaction time is shorter than the temperature, the mass transfer is weak, the contact between the catalyst and oxygen and lignocellulose is less, and the reaction effect is poor; above this temperature or above this reaction time, some of the products formed (mainly monolignol and hemicellulose degradation products) are susceptible to further degradation, resulting in losses and inhibition of subsequent enzymatic hydrolysis.
In order to improve the subsequent enzymolysis efficiency, the pretreated product needs to be subjected to purification treatment before enzymolysis. In a preferred embodiment, after the temperature-raising oxidation treatment and before the pretreatment product is obtained, the pretreatment method further comprises: performing water bath cooling on the product subjected to temperature rise and oxidation treatment, preferably performing ice water bath cooling to obtain a cooled product; and filtering and washing the cooled product to obtain a pretreated product.
Specifically, after the temperature rise oxidation reaction is finished, the temperature of a reaction product is high, and the rapid temperature reduction can be realized by adopting an ice water bath. Then, the oxidation product is subjected to solid-liquid separation, and solid residues in the oxidation product are washed to be neutral by deionized water, so that the pretreatment process is completed.
In a preferred embodiment, the lignocellulosic feedstock has a mass volume concentration of 5% to 20%, preferably 5% to 15%. The above-mentioned lignocellulosic raw material is used as a substrate for the reaction, i.e., the lignocellulosic raw material used for the reaction. 5 to 20% means the volume percentage of the lignocellulosic solid to the liquid (reaction solution), for example 10% means that 10g of the solid is reacted in 100mL of the reaction solution. The reaction solution is a solvent for reaction, and for example, the reaction solution can be a sodium phosphate solution prepared by using deionized water. Controlling the volume concentration within this range can promote contact of the alkaline salt catalyst with the lignocellulose, enhancing the beneficial effects of mass and heat transfer.
To further enhance the swelling effect of the alkaline salt solution on the lignocellulosic feedstock, in a preferred embodiment, prior to step S1, the pretreatment method further comprises: the lignocellulosic feedstock is comminuted into particles, preferably having a particle size of from 0.1cm to 1.5cm, more preferably from 0.3cm to 1.5 cm. The wood fiber raw material is crushed to reduce the crystallinity, reduce the particle size and increase the contact area between the raw material and the alkaline salt solution, thereby correspondingly improving the swelling and decomposing speed and efficiency. However, considering that the raw material having a relatively small particle size is difficult to recover after pretreatment, the particle size of the lignocellulosic raw material is preferably in the range of 0.3cm to 1.5 cm.
In a preferred embodiment, the solid-liquid mass ratio of the lignocellulosic feedstock to the alkaline salt solution is: 1: 5-1: 20, preferably, the temperature of the swelling treatment is 30-50 ℃, and more preferably, the time of the swelling treatment is 1-4 h. In the solid-liquid ratio, the decomposition and removal efficiency of lignocellulose of the same mass of lignocellulose raw materials is gradually increased along with the increase of alkaline salt solution, the solid recovery rate of the treated sample is also gradually increased, but the increasing speed begins to slow down after the solid-liquid mass ratio reaches 1:10, a plateau period is basically carried out after the decomposition and removal efficiency of the lignocellulose is increased after the solid-liquid mass ratio reaches 1:20, and the solid recovery rate of the treated sample is also obviously slowed down. The swelling treatment is carried out at the temperature of 30-50 ℃, the longer the swelling treatment time is, the more thoroughly the decomposition of the lignocellulose is relatively carried out, but the longer the swelling treatment time is, the decomposition efficiency can not be correspondingly improved any more.
Compared with the traditional alkaline pretreatment reagent, such as sodium hydroxide alkaline solvent or ammonia and other related solvents, the alkaline salt solution has the advantages of small corrosion, recoverability, small harm to the environment and the like. In addition, the alkaline salt pretreatment can reduce the generation of inhibitors while effectively removing lignin, thereby being more beneficial to the subsequent enzymatic hydrolysis process. In a preferred embodiment, the basic salt solution is a solution of a weakly basic salt; preferably, the weakly basic salt is sodium phosphate, sodium acetate, sodium carbonate, sodium sulfide, sodium hypochlorite or sodium silicate.
In a preferred embodiment, the concentration of the alkaline salt solution is 2 to 20 wt% (ratio of the mass of the alkaline salt to the total mass of the reaction solution, the total mass of the reaction also including the mass of the deionized water and the mass of the alkaline salt added, such as sodium phosphate). Controlling the concentration of the alkaline salt solution within the range has good swelling effect on the lignocellulose raw material.
In the application, the lignocellulose raw material is corn stalk, rice straw, wheat straw, spring bamboo shoot skin or bagasse. Agricultural wastes such as corn straws, bagasse or spring bamboo shoot skins which are the most extensive in source and the least expensive are preferably selected.
The concentration of the alkalescent alkaline salt solvent is 2-20% (mass fraction) of the total mass of the reaction solution; and introducing oxygen of 0.2-3.0 MPa into the closed high-pressure reaction kettle and maintaining the pressure. Higher alkaline salt solvent concentrations and higher oxygen contents are theoretically possible, but higher alkaline salt solvent concentrations and higher oxygen contents would increase production costs. The concentration of the alkaline salt solvent is preferably in the range of 2 to 15 percent (mass fraction), and the oxygen content is preferably in the range of 0.2 to 2.0 MPa. The reason that the oxygen pressure is preferably 0.2-2 MPa is to consider the influence of the increase of the oxygen pressure on the cost on one hand, and to consider that the oxygen pressure of 3MPa is easy to cause higher system pressure at high temperature, so that the requirement on the pressure resistance of reaction equipment is improved, and the method is not favorable for large-scale production.
After being processed by the pretreatment method, the lignocellulose has the following characteristics:
(1) the solid recovery rate of the pretreated lignocellulose raw material is between 45 and 75 percent.
(2) The lignin removal rate of the pretreated lignocellulose raw material is between 70 and 98 percent.
(3) The cellulose recovery rate of the pretreated lignocellulose raw material is between 80 and 90 percent.
(4) The hemicellulose removal rate of the pretreated lignocellulose raw material is between 10 and 50 percent.
After the lignocellulose raw material is treated by the pretreatment method, the lignocellulose raw material can be directly subjected to enzymatic hydrolysis saccharification, and in a preferred embodiment, complex enzyme is directly adopted to be hydrolyzed for 72 hours at 50 ℃ and pH4.8 in acetic acid-sodium acetate buffer solution to obtain various monosaccharides.
Accordingly, in a second exemplary embodiment of the present application, there is also provided a method of enzymatic digestion of lignocellulosic feedstock, the method comprising: pretreating the lignocellulose raw material by any one of the pretreatment methods to obtain a pretreated product; the pretreated product is subjected to enzymatic hydrolysis to obtain reducing sugar (monosaccharide such as glucose is mainly contained in the enzymatic hydrolysis liquid, but a small amount of cellobiose may be contained in the enzymatic hydrolysis liquid, and both monosaccharide and cellobiose belong to reducing sugar). The pretreatment method has high removal efficiency of lignocellulose and hemicellulose in the lignocellulose raw material, and relatively high retention of cellulose content, so that the enzymolysis saccharification efficiency and the reducing sugar yield of the enzymolysis method are improved.
In order to further improve the enzymolysis efficiency, in a preferred embodiment, the pretreated product is subjected to enzymolysis by using a complex enzyme, the complex enzyme is a mixture of cellulase and xylanase, and preferably, in the complex enzyme, the enzyme activity ratio of the cellulase to the xylanase in unit volume is as follows: the cellulase is xylanase, namely 1FPU:1FXU, wherein the FPU is a filter paper enzyme activity unit of the cellulase, and the FXU is an enzyme activity unit of the xylanase; more preferably, the amount of the complex enzyme is 10FPU/(g substrate) to 30FPU/(g substrate).
Wherein, FPU and FXU are respectively:
(1) FPU is the filter paper enzyme activity unit of cellulase, and one FPU unit represents the enzyme amount required by 1 × 6cm (50mg) filter paper as a substrate, and the cellulase can generate 1 μmol glucose within 1min at 50 deg.C and pH 4.8.
(2) FXU is the enzyme activity unit of xylanase, and one FXU unit represents the enzyme amount required by xylanase for producing 1 mu mol of xylose at pH4.8 and 50 ℃ within 1min by using xylan as a substrate.
In a preferred embodiment, the enzymatic hydrolysis is carried out at 50 ℃ under an acetic acid-sodium acetate buffer with pH4.8, preferably for 72-96 h.
Considering the factors of enzyme hydrolysis effect and cost comprehensively, the preferable dosage of the compound enzyme is 10FPU/(g substrate) -30 FPU/(g substrate), and the compound enzyme has the advantages of good enzyme hydrolysis effect and relatively low cost in the dosage range.
By the pretreatment method and the compound enzyme hydrolysis method, the yield of the enzymatic hydrolysis reducing sugar of the lignocellulose raw material is between 60 and 95 percent, and the yield of the optimized reducing sugar is over 85 percent.
The invention can utilize lignocellulose raw material to produce fuel ethanol or other biomass platform compounds taking saccharides as raw materials. The invention adopts agricultural wastes (such as straws and rice straws) as raw materials, the reaction reagent can be recycled for a plurality of times, the economic and environmental protection performance is good, the industrial production can be realized by utilizing the existing reaction equipment and device, and the invention has extremely high economic and social values for protecting the environment and recycling resources.
The advantageous effects of the present application will be further described with reference to specific examples.
Reducing sugar yield (%) ═ reducing sugar concentration x 0.9 x 100/enzyme hydrolysis substrate concentration;
glucose yield (%) ═ glucose concentration × 0.9 × 100/enzyme hydrolysis substrate concentration;
solids recovery (%) — mass of solids recovered x 100/mass of starting solids;
lignin removal rate (%) (1-lignin content in solids after pretreatment × (solid recovery) × 100/lignin content in raw material);
hemicellulose removal rate (%) (1-hemicellulose content in pretreated solids × (solid recovery rate) × 100/hemicellulose content in the raw material);
cellulose recovery (%). cellulose content in solids after pretreatment · solids recovery x 100/cellulose content in the feedstock.
Example 1: a pretreatment method and an enzyme hydrolysis method by using corn straws as raw materials.
Pulverizing corn stalk into granules smaller than 1cm, soaking, washing, and air drying. Cellulase (enzyme CP, enzyme activity 110FPU/mL) and Xylanase (Multifect Xylanase, 1600FXU/mL), both purchased from Sigma (St. Louis, Mo.). Preparing cellulase and xylanase into mixed enzyme solution by using acetic acid-sodium acetate buffer solution (pH4.8) and the enzyme activity ratio of the cellulase to the xylanase is 1FPU to 1FXU, and calculating the enzyme activity of the mixed enzyme solution by using the enzyme activity of filter paper.
The pretreatment process comprises the following steps:
weighing 3g of dried corn straw with the particle size of less than 1cm in a beaker, and adding 60g of Na with the concentration of 10 percent (mass fraction)3PO4Sealing the solution and the preservative film, and soaking for 4 hours at the temperature of 30 ℃. Pouring the soaked corn straws into a 100mL high-temperature high-pressure reaction kettle, introducing 1MPa oxygen, heating to 160 ℃ within 5 minutes, preserving heat for 1h, cooling for 10min in an ice-water bath after the reaction is finished, filtering and washing with deionized water, drying solid residues, and sealing bags for storage.
And (3) an enzymatic hydrolysis process:
0.2g of treated and untreated samples were placed in a 25mL Erlenmeyer flask, and 30FPU/g of substrate mixed with the enzyme solution was added, supplemented with a certain amount of pH4.8 acetate-sodium acetate buffer, to give a final substrate concentration for enzymatic hydrolysis of 20 g/L. The Erlenmeyer flask was placed in a constant temperature shaker (DHZ-052D) and treated with shaking at 160r/min at 50 ℃ for 72 h. And after the hydrolysis is finished, centrifuging for 10min at 8000r/min, and measuring reducing sugar by using a DNS method and measuring the content of glucose by using HPLC.
The pretreatment of the corn straws occurs under the condition of different substrate concentrations, and the solid recovery rate, the reducing sugar and glucose yield and the lignin removal rate of the treated samples are shown in figure 1. The sample under the optimum treatment conditions had a solids recovery mass of 1.58g, a lignin content of 1.5%, a hemicellulose content of 27.2%, and a cellulose content of 69.6%, and the solids recovery was 52.7%, the lignin removal rate was 95.4%, the cellulose recovery rate was 83.9%, and the hemicellulose removal rate was 40.2%. The corn stalk material was subjected to pretreatment and enzymatic hydrolysis for material analysis, and the results are shown in fig. 2. After pretreatment and enzyme hydrolysis, the final reducing sugar yield and glucose yield of the sample are respectively 90.9% and 61.2%, and are 74% higher than the yield of the untreated corn straw reducing sugar. As shown in FIG. 5 of a Scanning Electron Microscope (SEM) before and after sodium phosphate pretreatment of corn stalks, the untreated corn stalks are found to have smooth surfaces, and the treated corn stalks have rough and uneven surfaces, loose stalks and more pores.
Example 2: a pretreatment method and an enzyme hydrolysis method by using wheat straws as raw materials.
The wheat straw was pulverized into particles having a particle size of less than 0.5cm for use, and the mixed enzyme solution was the same as in example 1.
Weighing 3g of wheat straw particles, putting the wheat straw particles into a beaker, and adding 15g of Na with the concentration of 2 percent (mass fraction)2And sealing the S solution with a preservative film, and soaking for 1h at the temperature of 40 ℃. Pouring the soaked wheat straws into a 100mL high-temperature high-pressure reaction kettle, introducing 0.2MPa oxygen, heating to 100 ℃ within 5 minutes, preserving heat for 0.3h, cooling for 10min in an ice water bath after the reaction is finished, filtering and washing with deionized water, drying the solid residues, and sealing bags for storage. The enzymatic hydrolysis process was the same as in example 1, with 20FPU/(g substrate) of complex enzyme. The solid recovery mass of the pretreated sample was 2.1g, the lignin content was 5.1%, the hemicellulose content was 37.3%, and the cellulose content was 44.2%, and the solid recovery rate was 70.5%, the cellulose recovery rate was 82.6%, the hemicellulose removal rate was 13.2%, the lignin removal rate was 73.2%, and the final reducing sugar yield and glucose yield of the above sample after enzymatic hydrolysis were 61.6% and 37.2%, respectively.
Example 3: a pretreatment method and an enzyme hydrolysis method by using corn straws as raw materials.
The corn straws are crushed into particles with the particle sizes of 0.3 cm-0.5 cm, 0.5 cm-0.8 cm and 0.8 cm-1.2 cm for standby, and the mixed enzyme solution is the same as that in the embodiment 1.
Respectively weighing 3g of corn straw particles with different particle sizes in a beaker, adding 45g of Na with the concentration of 15 percent (mass fraction)3PO4Sealing the solution and preservative film, and soaking for 4h at 30 ℃. Pouring the soaked straws into a 100mL high-temperature high-pressure reaction kettle, introducing 1MPa oxygen, raising the temperature to 120 ℃ within 5 minutes, and preserving the temperature for 1h, wherein the rest steps are the same as those in the example 1. The results are shown in FIG. 3.
The results of comparing the reaction effects when 2g, 3g, 4g and 5g of the samples were added to a 100mL reaction vessel while changing the amount of the substrate to be loaded and the remaining reaction conditions were unchanged are shown in FIG. 4.
The loading capacity after pretreatment was 3g, the solid recovery mass of a sample having a particle size of 0.3cm to 0.5cm was 1.5g, the lignin content was 1.9%, the hemicellulose content was 25.4%, and the cellulose content was 71.5%, then the solid recovery rate was 50.3%, the cellulose recovery rate was 82.3%, the hemicellulose removal rate was 46.7%, the lignin removal rate was 94.5%, and the final reducing sugar yield and glucose yield of the above sample after enzymatic hydrolysis were 86.5% and 56.6%, respectively.
Example 4: a pretreatment method and an enzymatic hydrolysis method by taking spring bamboo shoot skin as a raw material.
The spring bamboo shoot skin is crushed into particles with the particle size of about 0.1cm for standby, and the mixed enzyme solution is the same as that in the embodiment 1.
Weighing 3g of spring bamboo shoot skin particles, putting the spring bamboo shoot skin particles into a beaker, adding 45g of NaClO solution with the concentration of 20% (mass fraction), sealing with a preservative film, and soaking for 1h at the temperature of 50 ℃. Pouring the soaked spring bamboo shoot skin into a 100mL high-temperature high-pressure reaction kettle, introducing 0.7Mpa of oxygen, heating to 150 ℃ within 5 minutes, preserving heat for 0.5h, cooling for 10min in an ice water bath after the reaction is finished, filtering and washing with deionized water, drying the solid residues, and sealing bags for storage. The enzymatic hydrolysis process was the same as in example 1. The solid recovery mass of the pretreated sample was 1.4g, the lignin content was 1.2%, the hemicellulose content was 34.6%, and the cellulose content was 54.1%, and the solid recovery rate was 46.2%, the cellulose recovery rate was 81.5%, the hemicellulose removal rate was 42.3%, the lignin removal rate was 96.2%, and the final reducing sugar yield and glucose yield of the above sample after enzymatic hydrolysis were 89.6% and 58.1%, respectively.
Example 5: a pretreatment method and an enzyme hydrolysis method by taking straws as raw materials.
The straw was pulverized into particles having a particle size of less than 1.5cm, and the enzyme mixture was the same as in example 1.
Weighing 3g of straw particles, placing the straw particles into a beaker, and adding 60g of Na with the concentration of 8 percent (mass fraction)3PO4Sealing the solution with preservative film, and placing at 35 deg.CSoaking for 2h under the environment. Pouring the soaked straws into a 100mL high-temperature high-pressure reaction kettle, introducing 3Mpa oxygen, heating to 100 ℃ within 5 minutes, preserving heat for 2 hours, cooling for 10 minutes in an ice-water bath after the reaction is finished, filtering and washing with deionized water, drying the solid residues, and sealing bags for storage. The enzymatic hydrolysis process was the same as in example 1. The solid recovery mass of the pretreated sample was 1.8g, the lignin content was 2.6%, the hemicellulose content was 31.3%, and the cellulose content was 55.7%, the solid recovery was 59.7%, the cellulose recovery was 87.9%, the hemicellulose removal was 38.2%, the lignin removal was 88.4%, and the final reducing sugar yield and glucose yield of the above sample after enzymatic hydrolysis were 83.8% and 51.6%, respectively.
Comparative example 1 and example 6:
washing and air drying the corn straws, and crushing the corn straws to be less than 0.5cm for later use. The mixed enzyme solution was the same as in example 1.
Examples of pretreatment with alkaline salt combined with aqueous hydrogen peroxide published in 2015:
weighing 1g of air-dried corn straw, placing the air-dried corn straw into a round-bottom flask, and adding 5mL of mixed solvent, wherein the mixed solvent is 10% H2O2And 20% Na3PO4Stirring and reacting for 1h at 120 ℃, adding deionized water with 2 times of volume after the reaction is finished and stirring for 30min at room temperature to regenerate the cellulose raw material. Then filtered and washed, and the solid residue is put into a sealed plastic bag for standby. The enzymatic hydrolysis process was the same as in example 1 above. The final reducing sugar yield and the glucose yield were 70.71% and 51.01%, respectively. Under the conditions, the solid recovery rate was 54.2%, the lignin removal rate was 84.11%, the cellulose recovery rate was 83.85%, and the hemicellulose removal rate was 35.5%.
Using 1MPa of O2Replacement of 10% H2O2The corn stalks are mixed with 10 percent of Na3PO4After soaking for 1h, the reaction is carried out for 1h at 120 ℃. The enzymatic hydrolysis process was the same as in example 1. The final reducing sugar yield and the glucose yield were 78.1% and 55.4%, respectively. Under this condition, the solid recovery rate of the sample was 55.7%, the lignin removal rate was 92.9%, the cellulose recovery rate was 85.7%, and the hemicellulose removal rate was 36.9%.
From the practical aspect, the method can reduce the use concentration of the alkaline salt, the treatment with hydrogen peroxide and 20% of sodium phosphate is needed in the comparative example, only 10% of sodium phosphate is needed in the example of the application, and the method is more advantageous in the aspects of reducing sugar yield, lignin removal rate and the like.
Comparative example 2 and example 7
The corn stalks are crushed into particles with the particle size of about 0.5cm for standby, and the mixed enzyme solution is the same as that in the embodiment 1.
Weighing 1g of corn straw particles, putting the corn straw particles into a beaker, and adding 5g of Na with the concentration of 1.5 percent (mass fraction)3PO4Sealing the solution and preservative film, and soaking for 0.5h at 30 ℃. Pouring the soaked corn straws into a 100mL high-temperature high-pressure reaction kettle, heating to 80 ℃ within 5 minutes, preserving heat for 0.5h, cooling for 10min in an ice-water bath after the reaction is finished, filtering and washing with deionized water, drying solid residues, and bagging for storage. The enzymatic hydrolysis process was the same as in example 1. The final reducing sugar yield and the glucose yield were 54.8% and 32.1%, respectively. Under this condition, the solids recovery of the sample was 77.2%, the lignin removal was 74.5%, the cellulose recovery was 90.4%, and the hemicellulose removal was 5.1%.
Weighing 1g of corn straw particles, putting the corn straw particles into a beaker, and adding 10g of Na with the concentration of 7 percent (mass fraction)3PO4Sealing the solution and preservative film, and soaking for 1h at 50 ℃. Pouring the soaked corn straws into a 100mL high-temperature high-pressure reaction kettle, introducing 0.5Mpa of oxygen, heating to 140 ℃ within 5 minutes, preserving heat for 0.5h, cooling for 10min in an ice water bath after the reaction is finished, filtering and washing with deionized water, drying solid residues, and sealing bags for storage. The enzymatic hydrolysis process was the same as in example 1. The final reducing sugar yield and the glucose yield were 83.8% and 55.4%, respectively. Under this condition, the solids recovery of the sample was 52.0%, the lignin removal was 92.2%, the cellulose recovery was 81.6%, and the hemicellulose removal was 42.5%.
Comparative example 3 and example 8
Washing and air drying the spring bamboo shoot skin and the corn straw, and crushing to be less than 0.5cm for later use. The mixed enzyme solution was the same as in example 1.
Examples of pretreatment with alkaline salt combined with aqueous hydrogen peroxide published in 2015:
weighing 1g of air-dried spring bamboo shoot skin, putting the air-dried spring bamboo shoot skin into a round-bottom flask, and adding 10mL of mixed solvent (namely the substrate concentration is 10g/L), wherein the mixed solvent is 5% H2O2And 9% Na3PO4Stirring to react for 6h at 50 ℃, adding equal volume of deionized water after the reaction is finished, and stirring for 30min at room temperature to regenerate the cellulose raw material. Then filtered and washed, and the solid residue is put into a sealed plastic bag for standby. The enzymatic hydrolysis process was the same as in example 1. The final reducing sugar yield and the glucose yield were 76.4% and 52.8%, respectively. Under the condition, the solid recovery rate is 42.2%, the lignin removal rate is 92.1%, the cellulose retention rate is 85.1%, and the hemicellulose removal rate is 37.4%.
Weighing 1g of corn straw, putting the corn straw into a beaker, and adding 15g of Na with the concentration of 8 percent (mass fraction)3PO4Sealing the solution and preservative film, and soaking for 3h at 40 ℃. Pouring the soaked corn straws into a 100mL high-temperature high-pressure reaction kettle, introducing 0.8Mpa of oxygen, heating to 140 ℃ within 5 minutes, preserving heat for 0.7h, cooling for 10min in an ice water bath after the reaction is finished, filtering and washing with deionized water, drying solid residues, and sealing bags for storage. The enzymatic hydrolysis process was the same as in example 1. The final reducing sugar yield and the glucose yield were 91.2% and 62.3%, respectively. Under this condition, the solid recovery rate of the sample was 52.1%, the lignin removal rate was 95.4%, the cellulose retention rate was 83.3%, and the hemicellulose removal rate was 40.7%.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: according to the pretreatment method, the lignocellulose raw material is subjected to swelling treatment by adopting the alkaline salt solution, the swollen lignocellulose is soaked, then the swelling product is placed in an oxygen environment for oxidation treatment, so that most of lignin and part of hemicellulose are removed under the condition of keeping more cellulose, namely, the decomposition efficiency of the pretreatment on the lignocellulose and hemicellulose is improved, further the subsequent enzymolysis is facilitated, and the enzymolysis saccharification efficiency and the reducing sugar yield are greatly improved.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (13)
1. A method of pretreating a lignocellulosic feedstock, comprising:
step S1, swelling the lignocellulose raw material by using an alkaline salt solution to obtain a swelling product;
and step S2, introducing oxygen into the swelling product for oxidation treatment to obtain a pretreated product.
2. The preprocessing method according to claim 1, wherein the step S2 includes:
step S21, placing the swelling product in a closed reaction kettle, introducing oxygen into the closed reaction kettle, and controlling the pressure of the introduced oxygen to be 0.2-3.0 MPa, preferably 0.2-2.0 MPa;
and step S22, heating and oxidizing the swelling product to obtain the pretreated product.
3. The pretreatment method according to claim 2, wherein the temperature-increasing oxidation treatment comprises:
and raising the temperature of the swelling product to 100-160 ℃, and carrying out oxidation treatment for 0.3-2 h at the temperature.
4. The pretreatment method according to claim 2 or 3, wherein after the temperature-raising oxidation treatment and before obtaining the pretreatment product, the pretreatment method further comprises:
carrying out water bath cooling on the product subjected to temperature rise and oxidation treatment, preferably carrying out ice water bath cooling to obtain a cooled product;
and filtering and washing the cooling product to obtain the pretreatment product.
5. The pretreatment method according to claim 1, wherein the mass volume concentration of the lignocellulosic feedstock is 5% to 20% of the substrate concentration.
6. The preprocessing method as claimed in claim 1, wherein before the step S1, the preprocessing method further comprises: the lignocellulosic feedstock is comminuted to particles, preferably having a particle size of from 0.1cm to 1.5cm, more preferably from 0.3cm to 1.5 cm.
7. The pretreatment method according to claim 1, wherein the solid-liquid mass ratio of the lignocellulosic feedstock to the alkaline salt solution is: 1: 5-1: 20, preferably, the temperature of the swelling treatment is 30-50 ℃, and more preferably, the time of the swelling treatment is 1-4 h.
8. The pretreatment method according to claim 1, wherein the basic salt solution is a solution of a weakly basic salt; preferably, the weakly basic salt is sodium phosphate, sodium acetate, sodium carbonate, sodium sulfide, sodium hypochlorite or sodium silicate.
9. The pretreatment method of claim 8, wherein the alkaline salt solution has a concentration of 2 wt% to 20 wt%.
10. The pretreatment method according to claim 1, wherein the lignocellulosic feedstock is corn stover, rice straw, wheat straw, spring bamboo bark, or bagasse.
11. An enzymatic hydrolysis method of a lignocellulosic feedstock, the enzymatic hydrolysis method comprising:
pretreating the lignocellulosic feedstock with the pretreatment method of any one of claims 1 to 10 to obtain the pretreated product;
and carrying out enzymolysis on the pretreated product to obtain reducing sugar.
12. The enzymolysis method according to claim 11, characterized in that the pretreated product is subjected to enzymolysis by using a complex enzyme, the complex enzyme is a mixture of cellulase and xylanase,
preferably, in the complex enzyme, the enzyme activity ratio of the cellulase to the xylanase in unit volume is as follows: the cellulase is xylanase, namely 1FPU:1FXU, wherein the FPU is a filter paper enzyme activity unit of the cellulase, and the FXU is an enzyme activity unit of the xylanase;
more preferably, the dosage of the compound enzyme is 10FPU/(g substrate) -30 FPU/(g substrate).
13. The enzymatic hydrolysis method according to claim 11, wherein the enzymatic hydrolysis is carried out under an acetic acid-sodium acetate buffer solution with a pH value of 4.8 at 50 ℃, and preferably the enzymatic hydrolysis time is 72-96 h.
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